Injectable shear-thinning hydrogel containing polypeptide therapeutic agent for enhanced tumor therapy

ABSTRACT

We have developed novel shear-thinning biomaterials using silica nanoparticles, gelatin-based polymers and polypeptides such as anti-PD-1 antibodies. Shear-thinning biomaterial technology offers enables polymers and drugs loaded inside such polymers to be easily delivered directly through catheters into target area for use, for example, in cancer therapy and immunotherapy. When a force above a certain threshold is applied to inject such materials, they “thin” and behaves as a semi-solid, allowing the material to readily flow through a catheter. When the force is removed, the material instantly becomes a soft solid with significant cohesive properties that prevent it from dislodging or breaking up.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 63/250,441 filed on Sep. 30, 2021 and U.S.Provisional Patent Application Ser. No. 63/249,949 filed on Sep. 29,2021. These provisional applications are incorporated herein byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under Grant NumbersHL140951, HL137193, awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

TECHNICAL FIELD

The present invention relates to shear thinning biomaterials comprisingbioactive agents and methods for making and using them.

BACKGROUND OF THE INVENTION

Shear-thinning hydrogels are non-Newtonian materials that behave asviscous fluids under shear stress and then recover solid-like propertiesupon elimination of the stress [1]. Due to these properties, injectableshear-thinning biomaterials (STB) are attracting attention as a group ofself-healing materials that allow for fluent infusion and localequilibrium after approaching the final application site. In clinicalapplications, STBs can be delivered into the body using a needle or ageneral/microcatheters by manual pressure [2-4]. To optimize theclinical application, it is necessary to adjust the physical propertiesof STB according to the specific clinical situations. A number ofphysiological and clinical parameters affect the formulation design,including but not limited to (1) peri-tumoral pH (environmental pH oftumor) (2) Tumor cell density, (3) Extracellular matrix synthesis ofTumor, (4) Treatment duration, (5) Aggressiveness of tumor, (6) Tumorphysiology including central necrotic lesion formation, (7)Accessibility of tumor (8), Drug solubility, and (9) Hydrophilicity ofdrugs/agents. By changing the physical properties, one can synthesizetailored hydrogels for specific clinical applications, such asembolizing a certain size of blood vessel, controlled drug release, andmodulation of the stiffness of tissue engineering scaffolds.

The physical properties of conventional STBs can be modulated by acombination of several carbon-based, polymeric, and inorganicnanomaterials [5-8]. Several biomaterials, such as gelatin, hyaluronicacid, chitosan, collagen, and alginate have been previously used alongwith inorganic constituents to form STBs [14-17]. In particular, gelatinlimits the adsorption of nonspecific proteins, enhanced hemolysis, andultimately prolongs clotting time, demonstrating substantially improvedhemocompatibility of STB in vitro [18]. Furthermore, the application ofgelatin in tissue engineering and regenerative medicine has beenapproved by the Food and Drug Administration (FDA) [19-21]. ConventionalSTBs are prepared by mixing gelatin with synthetic clay nanoparticles,LAPONITE®, for hemostasis and endovascular embolization [18, 22]. TheseSTBs exhibit strong shear-thinning behavior as well as biocompatibleproperties ranging from blood coagulation to minimized inflammatoryresponse. Others have extended this work to implement STBs as embolicagents [23], functionalized scaffolds [24, 25], 3D-bioinks [26] and drugdelivery systems [27]. Unfortunately, however, synthetic claynanoparticles such as LAPONITE are crystallized nanoparticles and thesize, surface chemistry are not easily tuned. In addition, thebiocompatibility of LAPONITE in various in vivo uses needs to be furtherinvestigated, a fact which complicates its use in clinical applications.

For the reasons noted above, there is a need in the art for newshear-thinning materials and methods for making and using them.

SUMMARY OF THE INVENTION

As discussed below, we have developed an immune checkpoint inhibitor(ICI) loaded injectable shear thinning biomaterial (STB) using silicatenanoplatelet (LAPONITE XLG) and gelatin-based polymers. Injectable STBis one class of hydrogels which can be injected by application of shearstress during injection and rapidly self-healing after removal ofstress. STBs have attracted considerable attention in biomedical fields,especially for delivery of drugs and cells and for endovascularembolization in a minimally invasive manner due to its ability to beeasily applied through a syringe and undergo a fast sol-gel transitionat the target sites. LAPONITE (LAPONITE XLG) is a synthetic disk-likesilicate nanoparticles with a diameter of 25 nm and a thickness of about1 nm. The surfaces of LAPONITE are negatively charged while their slicesare positively charged. These unique characteristics not only allow thisclay to form a hydrogel with shear-thinning property but also endow thempromising potential to deliver various drugs by adsorbing them throughthe electrostatic interaction. Herein, we described a strategy toutilize the injectable STB containing LAPONITE and gelatin to enhancetumor therapy by combining checkpoint blockade and endovascularembolization. As shown in Scheme 1 (FIG. 4 ), PD-1-blocking antibody(anti-PD-1) molecules are firstly adsorbed on the surfaces of LAPONITEthrough electrostatic interaction, which subsequently delivered tovasculature near tumor through syringe in the form of STBs. Anti-PD-1are released from STBs and then enter the interior of tumor.

The invention disclosed herein has a number of embodiments. Embodimentsof the invention include, for example, a composition of mattercomprising a gelatin, silicate nanoparticles and a polypeptide, which istypically a therapeutic agent such as an immune checkpoint inhibitorantibody (e.g. Anti-PD-1). In such compositions, amounts of the gelatin,the silicate nanoparticles and the polypeptide are selected to form ashear thinning hydrogel. In certain embodiments of the invention, thecomposition comprises from about 1% (w/w) to about 5% (w/w) gelatin(e.g., ranging anywhere from about 1% (w/w) to about 2% (w/w) to about3% (w/w) to about 4% (w/w) to about 5% (w/w) gelatin) (in other words,ranging between any two of the preceding numerical values), and fromabout 1% (w/w) to about 5% (w/w) silicate nanoparticles (e.g., ranginganywhere from about 1% (w/w) to about 2% (w/w) to about 3% (w/w) toabout 4% (w/w) to about 5% (w/w) silicate nanoparticles). In certainembodiments of the invention, the composition comprises from about 0.5%(w/w) to about 85% (w/w) gelatin and silicate nanoparticles (e.g.,ranging anywhere from about 0.5% (w/w) to about 1% (w/w) to about 2%(w/w) to about 5% (w/w) to about 10% (w/w) to about 25% (w/w) to about50% (w/w) to about 75% (w/w) to about 85% (w/w) gelatin and silicatenanoparticles). In certain embodiments of the invention, the ratio ofthe silicate nanoparticles to the gelatin is from about 1.0 to about 0.1(e.g., ranging anywhere from about 1.0 to about 0.9 to about 0.8 toabout 0.7 to about 0.6 to about 0.5 to about 0.4 to about 0.3 to about0.2 to about 0.1). In certain embodiments, the gelatin is methacrylatedgelatin (GelMA), acrylated gelatin, or thiolated gelatin. In certainembodiments of the invention, the composition comprises about 0.5% (w/w)to about 99% (w/w) water (e.g., ranging anywhere from about 0.5% (w/w)to about 1% (w/w) to about 2% (w/w) to about 5% (w/w) to about 10% (w/w)to about 25% (w/w) to about 50% (w/w) to about 75% (w/w) to about 90%(w/w) to about 95% (w/w) to about 97.5% (w/w) to about 99% (w/w) water).In certain embodiments, the composition comprises from about 0.01% (w/w)to about 20% (w/w) of the polypeptide therapeutic agent (e.g., ranginganywhere from about 0.01% (w/w) to about 0.02% (w/w) to about 0.5% (w/w)to about 1% (w/w) to about 2% (w/w) to about 5% (w/w) to about 10% (w/w)to about 15% (w/w) to about 20% (w/w) of the polypeptide therapeuticagent). In certain embodiments, the polypeptide therapeutic agent isselected from the group consisting of cytokines, antibodies, anticancerenzymes, tumor antigens, and pro-apoptotic proteins or peptides. Incertain embodiments, the polypeptide therapeutic agent is selected fromthe group consisting of IL-2, IL-12, IFN-γ, TNF-α, Trastuzumab,Pertuzumab, Bevacizumab, Rituximab, Atezolizumab, Durvalumab, Nivolumab,caspase-3, recombinase (Cre), L-asparaginase, RNase A, DNase I, cGAMPsynthase, granzyme B, catalase, OVA, TRP2, Hpg10025-33, p-AH1-A5,MAGE-A3, p53, cytochrome c, KLAKLAKKLAKLAKGG, PTEN, and Saporin.Typically, these compositions further comprise a pharmaceuticalexcipient selected from the group consisting of a preservative, atonicity adjusting agent, a detergent, a viscosity adjusting agent, asugar and a pH adjusting agent. In some embodiments of the invention,the composition further comprises a human cancer cell. The compositionsof the invention include one or more polypeptide therapeutic or otheragents. In illustrative embodiments of the invention, the therapeuticagent is anti-PD-1 antibody.

In some embodiments of the invention, amounts of the components are suchthat when disposed in an environment having a pH of 7.4, less than 10%or less than 5% of agent is released from the thinning hydrogel over aperiod of 15 days; and when disposed in in an environment having a pH of5.0, more than 5% or more than 10% is released from shear thinninghydrogel over a period of 15 days. In certain embodiments of theinvention, the shear thinning hydrogel requires an injection force of atleast 5 newtons but less than 30 newtons to extrude the shear thinninghydrogel from a 2.4Fr Catheter/1 cc syringe.

Another embodiment of the invention is a method of making ashear-thinning biocompatible composition disclosed herein comprisingcombining together spherical silica nanoparticles, gelatin and a smalltherapeutic molecule such as anti-PD-1 antibody, and optionally apharmaceutical excipient so as to form a shear-thinning biocompatiblecomposition. In certain embodiments of these methods, a surface propertyof the spherical silica nanoparticles, a median diameter of thespherical silica nanoparticles, a relative amount of spherical silicananoparticles; and/or a relative amount of gelatin or the like isselected to tune or modulate one or more rheological properties orpolypeptide release profile of the shear-thinning biocompatiblecomposition. By modulating the mechanical properties of the compositionsof the invention in this manner, embodiments of the invention can betailored for use in a variety of different clinical applications.

Another embodiment of the invention is a method of delivering ashear-thinning biocompatible composition disclosed herein to apreselected site (e.g. an in vivo location comprising cancer cells).Typically, such methods comprise disposing the composition in a vesselhaving a first end comprising an opening and a second end (e.g. acatheter); applying a force to the second end of the vessel, wherein theforce is sufficient to liquify the composition; and then delivering thecomposition out of the vessel through the opening and to the preselectedsite. In some embodiments, the vessel comprises a syringe loaded withthe composition, the syringe configured for fluid communication with aneedle and/or a catheter tube. Embodiments of such methods includetreatment regimens that use a shear-thinning biocompatible compositiondisclosed herein to deliver therapeutic polypeptides such as antibodies.In certain embodiments, the present disclosure is directed to method oftreating a solid tumor in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of ashear-thinning biocompatible composition disclose herein. In certainembodiments, the solid tumor is selected from a blood vessel tumor,brain tumor (e.g., meningioma or glioblastoma), spinal tumor, carotidbody tumor, liver cancer, lung cancer, neuroendocrine tumor, renaltumor, pancreatic tumor or prostatic tumor. In certain embodiments, thesolid tumor is selected from skin cancer (e.g., melanoma, mast celltumor, squamous cell carcinoma, or basal cell tumor), breast cancer,sarcomatous tumor (e.g., fibrosarcoma, leiomyosarcoma, rhabomyosarcoma,osteosarcoma, or chondrosarcoma), and lymphomatous tumor. In certainembodiments, the composition is administered to a localized area in needof treatment. In certain embodiments, the composition provides sustainedrelease of a therapeutically effective amount of the polypeptidetherapeutic agent to the localized area. In certain embodiments, thelocalized area comprises a solid tumor. In certain embodiments, thecomposition provides tumor embolization and sustained release of atherapeutically effective amount of the polypeptide therapeutic agent tothe tumor.

Other objects, features and advantages of the present invention willbecome apparent to those skilled in the art from the following detaileddescription. It is to be understood, however, that the detaileddescription and specific examples, while indicating some embodiments ofthe present invention, are given by way of illustration and notlimitation. Many changes and modifications within the scope of thepresent invention may be made without departing from the spirit thereof,and the invention includes all such modifications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows anti-PD-1 released from STHs with differentLaponite/gelatin ratios in PBS (pH=7.4). FIG. 1B and FIG. 1C showanti-PD-1 released from various compositions loaded with differentantibody concentrations in PBS (pH=7.4). FIG. 1D and FIG. 1E showrelease profile data from various compositions comprising anti-PD-1antibody loaded STBs under different pH conditions.

FIG. 2A and FIG. 2B shows shear stress data from various compositions ofSTBs with different antibody concentrations. FIG. 2C and FIG. 2D showthe mechanical properties of various compositions of STBs with differentantibody concentrations.

FIG. 3A shows photographs of tumors treated with embodiments of theinvention. FIG. 3B provides data from an in vivo study assessingdirect-contact anti-cancer effect of anti-PD-1 antibody loaded STBs.FIG. 3C shows data from a tumor weight study on the day of sacrifice.

FIG. 4 shows Scheme 1, an illustration of dual functions of injectableSTHs to enhance tumor therapy by combining the anti-PD-1 delivery andendovascular embolization.

DETAILED DESCRIPTION OF THE INVENTION

In the description of embodiments, reference may be made to theaccompanying figures which form a part hereof, and in which is shown byway of illustration a specific embodiment in which the invention may bepracticed. It is to be understood that other embodiments may beutilized, and structural changes may be made without departing from thescope of the present invention. Unless otherwise defined, all terms ofart, notations and other scientific terms or terminology used herein areintended to have the meanings commonly understood by those of skill inthe art to which this invention pertains. In some cases, terms withcommonly understood meanings are defined herein for clarity and/or forready reference, and the inclusion of such definitions herein should notnecessarily be construed to represent a substantial difference over whatis generally understood in the art. Many of the aspects of thetechniques and procedures described or referenced herein are wellunderstood and commonly employed by those skilled in the art. Thefollowing text discusses various embodiments of the invention.

Shear-thinning biomaterial (STB) technology offers unique propertiesenabling a solid polymer and drugs loaded inside to be easily delivereddirectly through means such as catheters into a target area. In view ofthis, we have developed a class of novel shear-thinning biomaterialsusing silica nanoparticles and gelatin-based polymers. Embodiments ofthe invention include, for example, a shear-thinning biocompatiblecomposition of matter comprising silica nanoparticles, gelatin and apolypeptide such as an antibody. Among inorganic composite compositions,silica is classified as “Generally Recognized As Safe (GRAS)” FDA, andis considered one of the most biocompatible materials. In view of this,silicate nanoparticles have been employed in pharmaceutical, cosmeticand food industries as active ingredients or rheological modifiers dueto their uniform particle size with electrical surface charges andbioactive properties [9, 10]. In addition, the size, structure, surfaceproperties of silica nanoparticles can be easily tuned, properties whichallow silica nanoparticles to provide a class of materials that areuseful in a wide variety of biomedical applications.

The compositions of the invention can include further constituents suchas additional polymers, excipients, therapeutic agents and the like. Forexample, compositions of the invention can include one or more Food andDrug Administration (FDA) approved or cytocompatible polymers. Suchpolymers include alginate, chitosan, collagen, hyaluronic acid (HA),chondroitin sulfate (ChS), dextrin, gelatin, fibrin, peptide, and silk.Synthetic polymers such as poly(ethylene glycol) (PEG), poly(ethyleneoxide) (PEO), poloxamer (Pluronic®) (PEO-PPO-PEO), polyoxamine(Tetronic®) (PEO-PPO), poly(vinyl alcohol) (PVA),poly(lactic-co-glycolic acid) (PLGA), poly(glycolic acid) (PGA),poly(lactic acid) (PLA), polycaprolactone (PCL), poly(L-glutamic acid)(PLga), polyanhydrides, poly(N-isopropylacrylamide) (PNIPAAm),polyaniline and the like can also be included in compositions of theinvention. As is known in the art, preparations of hydrogels can be madeto include either chemically or physically crosslinked materials.

Certain embodiments of the compositions of the invention include, forexample a pharmaceutical excipient such as one selected from the groupconsisting of a preservative, a tonicity adjusting agent, a detergent, aviscosity adjusting agent, a sugar and a pH adjusting agent. Forcompositions suitable for administration to humans, the term “excipient”is meant to include, but is not limited to, those ingredients describedin Remington: The Science and Practice of Pharmacy, Lippincott Williams& Wilkins, 21st ed. (2006) the contents of which are incorporated byreference herein.

Optionally, the compositions of the invention include one or morepolypeptide therapeutic agents such as an immune checkpoint inhibitor orthe like. In a working embodiment of the invention disclosed herein, thepolypeptide therapeutic agent is anti-PD-1 antibody. Compositions of theinvention can be formulated for use as carriers or scaffolds oftherapeutic agents such as drugs, cells, proteins, and bioactivemolecules (e.g., enzyme). As carriers, such compositions can incorporatethe agents and deliver them to a desired site in the body for thetreatments of a variety of pathological conditions. These include, forexample, infectious and inflammatory diseases (e.g. Parkinson's disease,bacterial and antimicrobial infection, diabetes and the like) as well ascancers (e.g. colon, lung, breast, ovarian, lymphoma cancers and thelike). In addition, as scaffolds, compositions of the invention canprovide a flexible dwelling space for cells and other agents for use intissue repair and the regeneration of desired tissues (e.g. forcartilage, bone, retina, brain, and, neural tissue repair, vascularregeneration, wound healing and the like). Moreover, embodiments of theinvention can include immunomodulatory agents useful for immunotherapyin order to, for example, modulate components of the immune system.Certain illustrative materials and methods that can be adapted for usein such embodiments of the invention are found, for example inHydrogels: Design, Synthesis and Application in Drug Delivery andRegenerative Medicine 1st Edition, Singh, Laverty and Donnelly Eds; andHydrogels in Biology and Medicine (Polymer Science and Technology) UKed. Edition by J. Michalek et al.

Embodiments of the invention include, for example, a composition ofmatter comprising a gelatin, silicate nanoparticles and a polypeptide.In such compositions, amounts of the gelatin, the silicate nanoparticlesand the polypeptide are selected to form a shear thinning hydrogel. Incertain embodiments of the invention, the composition comprises fromabout 1% to about 5% gelatin, and from about 1% to about 5% silicatenanoparticles. Typically, these compositions further comprise apharmaceutical excipient selected from the group consisting of apreservative, a tonicity adjusting agent, a detergent, a viscosityadjusting agent, a sugar and a pH adjusting agent. In some embodimentsof the invention, the composition further comprises a human cancer cell.In illustrative embodiments of the invention, the polypeptide is ananti-PD-1 antibody.

In some embodiments of the invention, the composition is disposed withina vessel (e.g. a catheter) selected for its ability to facilitate a usermodulating one or more rheological properties of the composition.Certain illustrative materials and methods that can be adapted for usein embodiments of the invention are found, for example in BiomedicalHydrogels: Biochemistry, Manufacture and Medical Applications (WoodheadPublishing Series in Biomaterials) 1st Edition; Steve Rimmer (Editor).As shown in FIGS. 2A-2D, illustrative embodiments of the invention, thecomponents of the composition are selected to provide desirablerheological properties.

Another embodiment of the invention is a method of delivering ashear-thinning biocompatible composition disclosed herein to apreselected site (e.g. an in vivo location where an individual hasexperienced trauma or injury or exhibits a pathology such as a cancer).Typically, such methods comprise disposing the composition in a vesselhaving a first end comprising an opening and a second end (e.g. acatheter); applying a force to the second end of the vessel, wherein theforce is sufficient to liquify the composition; and then delivering thecomposition out of the vessel through the opening and to the preselectedsite. Embodiments of such methods include treatment regimens that use ashear-thinning biocompatible composition disclosed herein to deliver atherapeutic agent as shown, for example, in FIGS. 3A-3C.

Yet another embodiment of the invention is a method of making ashear-thinning biocompatible composition disclosed herein comprisingcombining together silica nanoparticles and gelatin, a polypeptide (e.g.anti-PD-1 antibody) and optionally a pharmaceutical excipient so as toform a shear-thinning biocompatible composition. In certain embodimentsof these methods, a surface property of the silica nanoparticles, amedian diameter of the silica nanoparticles, a relative amount of silicananoparticles; and/or a relative amount of gelatin or the like isselected to tune or modulate one or more rheological properties orpolypeptide release profile properties of the shear-thinningbiocompatible composition. By modulating the mechanical properties ofthe compositions of the invention in this manner, embodiments of theinvention can be tailored for use in a variety of different clinicalapplications. In this context, a wide variety of art accepted materialsand methods can be adapted for use in embodiments of the invention, forexample those disclosed in U.S. Patent Publication Nos.: 20050227910,20100120149, 20120315265, 20140302051 and 20190290804; and Lee,Biomaterials Research volume 22, Article number: 27 (2018); Thambi etal., J Control Release. 2017 Dec. 10; 267:57-66. doi:10.1016/j.jconrel.2017.08.006. Epub 2017 Aug. 4; and Gianonni et al.,Biomater. Sci., 2016, the contents of which are incorporated byreference.

Illustrative Materials and Methods of the Invention

As an illustrative working embodiment of the invention, we developed ananti-PD-1 antibody loaded injectable shear thinning biomaterials (STB)using silicate nanoplatelet (LAPONITE XLG) and gelatin-based polymers.Shear-thinning biomaterial technology offers unique properties enablingdrugs loaded inside to be easily delivered directly through needles orcatheters into target area. We focused on the fact that shear thinningbiomaterial is 1) injectable and easy to apply, and 2) exhibits highlocalization due to the high mechanical stability after injection.Therefore, we loaded anti-cancer drug, anti-PD-1 antibody into the shearthinning material to treat solid tumor.

We tested three different STB compositions (Gelatin 4.5%/LAPONITE 1.5%[6NC25]; Gelatin 3.0%/LAPONITE 3.0% [6NC50]; Gelatin 1.5%/LAPONITE 4.5%[6NC75]) for the ICI-loading biomaterials and anti-PD-1 antibody releaseprofile was analyzed for 30 days (FIG. 1A). The release ratio ofanti-PD-1 from STBs was sharply decreased with the increase of LAPONITEcontent in the STBs. As shown in FIGS. 1B and 1C, when the antibodyconcentration was adjusted ranging from 0.5 μg/mg STB to 5.0 μg/mg STB,the release rate of anti-PD-1 was remarkably increased from 1 ug/mg STB(2.58±0.05%) to 5.0 ug/mg STB (8.92±0.14%) in the case of 6NC50 (FIG.1C). While the highest release of anti-PD-1 was observed at the antibodyconcentration of 1 ug/mg STB in the case of 6NC25 (FIG. 1B). PBS withdifferent pH values (7.4, 6.0, and 5.0) was used to evaluate itsinfluence on the release of anti-PD-1(FIGS. 1D and 1E). The results showthat the release of anti-PD-1 was greatly improved in the acidicconditions.

As shown in FIGS. 2A and 2B, the similar shear stress-shear rate curveswere observed in 6NC25 loading anti-PD-1 with different concentrations,indicating no significant influence on shear-thinning behavior of 6NC25and 6NC50 after loading of antibody at 37° C. Storage modulus reflectsthe recovery from liquid-like behavior to solid-like behavior afterapplication of high strain (100%) and low strain (1%). FIGS. 2C and 2Dshowed that storage moduli of STBs loading anti-PD-1 with differentconcentrations after five cycles of high (100%) to low (1%) oscillatorystrain amplitudes. In the case of 6NC25 and 6NC50, the storage modulusduring 100% strain oscillations was slightly lower than the initialvalues during high strain after five cycles, indicating rapid recoveryof the organic-inorganic hybrid network consisting of LAPONITE andgelatin. These results show that the both shear-thinning performance andstorage moduli of STBs remain stable under physiological temperatureregardless of loading anti-PD-1 with different concentrations.

Finally, in vivo anti-cancer efficacy of ICI-STB was analyzed in amelanoma tumor model made by subcutaneous injection of B16F10 melanomatumor cells into C57Bl/6J mice (FIG. 3A). In gross, STB (6NC25) itselfdid not show anti-cancer efficacy, while tumor size decreased inanti-PD1 loaded 6NC25. On the tumor growth curve measured by caliperevery two days, anti-PD1 loaded STB group was found to inhibit melanomagrowth (FIG. 3B). Interestingly, median tumor weights of ICI-STB(anti-PD1 loaded 6NC25) was less than others in tumor weight (FIG. 3C).

In summary, we developed a novel shear-thinning biomaterial with thecomposite of anti-PD-1 antibody, gelatin and biocompatible silicananoparticles with selected desirable properties. By tuning theproperties of silica nanoparticles such as particles size andcomposition of gelation/silica nanoparticles, the mechanical andrheological properties can be carefully adjusted to meet differentrequirements. All the compositions used in these experiments areinjectable through different sized catheters. The rheological testsshowed rapid recovery and mechanical stability with shear-thinningcharacteristics. Gelatin-Silica nanoparticles-based STBs displaysuperior biological stability, body temperature extrudability,shear-thinning behavior, and rapid network recoverability. Theseattractive physicochemical properties are favorable for easyadministration in vivo, and a gelatin-Silica nanoparticle-based STB mayhold great potential in drug delivery, endovascular embolization, tissueregeneration, bioprinting and for other biomedical applications.

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All publications mentioned herein (e.g. the references numericallylisted above, and U.S. Patent Publication 20180104059) are incorporatedherein by reference to disclose and describe aspects, methods and/ormaterials in connection with the cited publications.

The Appendix that is included with this provisional application providesinvention disclosure that is formatted for publication in a journal.This disclosure shows various aspects and embodiments of the invention.This Appendix is incorporated by reference herein.

1. A shear thinning hydrogel composition comprising: a gelatin; silicatenanoparticles; and a polypeptide therapeutic agent; wherein: thegelatin, the silicate nanoparticles and the therapeutic agent form ashear thinning hydrogel.
 2. The composition of claim 1, wherein thecomposition comprises: from about 1% (w/w) to about 5% (w/w) gelatin;and from about 1% (w/w) to about 5% (w/w) silicate nanoparticles.
 3. Thecomposition of claim 1, wherein when disposed in an environment having apH of 7.4, less than 10% or 5% of agent is released from the shearthinning composition over a period of 15 days; and when disposed in inan environment having a pH of 5.0, more than 5% or 10% is released fromshear thinning hydrogel over a period of 15 days.
 4. The composition ofclaim 1, wherein the composition comprises from about 0.5% (w/w) toabout 85% (w/w) gelatin and silicate nanoparticles.
 5. The compositionof claim 1, wherein the ratio of the silicate nanoparticles to thegelatin is from about 1.0 to about 0.1.
 6. The composition of claim 1,wherein the gelatin is methacrylated gelatin (GelMA), acrylated gelatin,or thiolated gelatin.
 7. The composition of claim 1, further comprisingabout 0.5% (w/w) to about 99% (w/w) water.
 8. The composition of claim1, wherein the composition comprises from about 0.01% (w/w) to about 20%(w/w) of the polypeptide therapeutic agent.
 9. The composition of claim1, wherein the polypeptide therapeutic agent is selected from the groupconsisting of cytokines, antibodies, anticancer enzymes, tumor antigens,and pro-apoptotic proteins or peptides.
 10. The composition of claim 9,wherein the polypeptide therapeutic agent is selected from the groupconsisting of IL-2, IL-12, IFN-γ, TNF-α, Trastuzumab, Pertuzumab,Bevacizumab, Rituximab, Atezolizumab, Durvalumab, Nivolumab, caspase-3,recombinase (Cre), L-asparaginase, RNase A, DNase I, cGAMP synthase,granzyme B, catalase, OVA, TRP2, Hpg10025-33, p-AH1-A5, MAGE-A3, p53,cytochrome c, KLAKLAKKLAKLAKGG, PTEN, and Saporin.
 11. The compositionof claim 1, wherein the polypeptide therapeutic agent comprises ananti-PD-1 antibody.
 12. The composition of claim 1, wherein the shearthinning hydrogel requires an injection force of at least 5 newtons butless than 30 newtons to extrude the shear thinning hydrogel from a 2.4FrCatheter/1 cc syringe.
 13. The composition of claim 1, furthercomprising a human cancer cell.
 14. The composition of claim 1, furthercomprising a pharmaceutical excipient selected from a preservative, atonicity adjusting agent, a detergent, a viscosity adjusting agent, asugar and a pH adjusting agent.
 15. The composition of claim 1, whereinthe composition is disposed within a catheter.
 16. A method ofdelivering a composition to a preselected site comprising: disposing thecomposition in a vessel having a first end comprising an opening and asecond end, the composition comprising a gelatin, silicatenanoparticles, and a polypeptide therapeutic agent, wherein the gelatin,the silicate nanoparticles and the therapeutic agent form a shearthinning hydrogel; applying a force to the second end of the vessel,wherein the force is sufficient to liquify the composition; deliveringthe composition out of the vessel through the opening and to thepreselected site.
 17. The method of claim 16, wherein the site is an invivo site.
 18. The method of claim 16, wherein the site is at an in vivolocation comprising cancerous cells.
 19. The method of claim 16, whereinthe vessel is a catheter.
 20. A method of treating a solid tumor in asubject in need thereof, comprising administering to the subject atherapeutically effective amount of a composition comprising a gelatin,silicate nanoparticles, and a polypeptide therapeutic agent, wherein thegelatin, the silicate nanoparticles and the therapeutic agent form ashear thinning hydrogel.